The Jun N-terminal kinase (JNK) pathway is activated by exposure of cells to environmental stress or by treatment of cells with pro-inflammatory cytokines. Targets of the JNK pathway include the transcription factors c-jun and ATF2 (Whitmarsh A. J., and Davis R. J. J. Mol. Med. 74:589-607, 1996). These transcription factors are members of the basic leucine zipper (bZIP) group that bind as homo- and hetero-dimeric complexes to AP1 and AP-1-like sites in the promoters of many genes (Karin M., Liu Z. G. and Zandi E. Curr Opin Cell Biol 9:240-246, 1997). JNK binds to the N-terminal region of c-jun and ATF-2 and phosphorylates two sites within the activation domain of each transcription factor (Hibi M., Lin A., Smeal T., Minden A., Karin M. Genes Dev. 7:2135-2148, 1993; Mohit A. A., Martin M. H., and Miller C. A. Neuron 14:67-75, 199). Three JNK enzymes have been identified as products of distinct genes (Hibi et al, supra; Mohit et al., supra). Ten different isoforms of JNK have been identified. These represent alternatively spliced forms of three different genes: JNK1, JNK2, and JNK3. JNK1 and 2 are ubiquitously expressed in human tissues, whereas JNK3 is selectively expressed in the brain, heart, and testis (Dong, C., Yang, D., Wysk, M., Whitmarsh, A., Davis, R., Flavell, R. Science 270:1-4, 1998). Gene transcripts are alternatively spliced to produce four-JNK1 isoforms, four-JNK2 isoforms, and two-JNK3 isoforms. JNK1 and 2 are expressed widely in mammalian tissues, whereas JNK3 is expressed almost exclusively in the brain. Selectivity of JNK signaling is achieved via specific interactions of JNK pathway components and by use of scaffold proteins that selectively bind multiple components of the signaling cascade. JIP-1 (JNK-interacting protein-1) selectively binds the MAPK module, MLK→JNKK1→JNK. It has no binding affinity for a variety of other MAPK cascade enzymes. Different scaffold proteins are likely to exist for other MAPK signaling cascades to preserve substrate specificity.
JNKs are activated by dual phosphorylation on Thr-183 and Tyr-185. JNKK1 (also known as MKK 4) and JNKK2 (MKK7), two MAPKK level enzymes, can mediate JNK activation in cells (Lin A., Minden A., Martinetto H., Claret F.-Z., Lange-Carter C., Mercurio F., Johnson G. L., and Karin M. Science 268:286-289, 1995; Tournier C., Whitmarsh A. J., Cavanagh J., Barrett T., and Davis R. J. Proc. Nat. Acad. Sci. USA 94:7337-7342, 1997). JNKK2 specifically phosphorylates JNK, whereas JNKK1 can also phosphorylate and activate p38. Both JNKK1 and JNKK2 are widely expressed in mammalian tissues. JNKK1 and JNKK2 are activated by the MAPKKK enzymes, MEKK1 and 2 (Lange-Carter C. A., Pleiman C. M., Gardner A. M., Blumer K. J., and Johnson G. L., Science, 260:315-319, 1993; Yan M., Dai J. C., Deak J. C., Kyriakis J. M., Zon L. I., Woodgett J. R., and Templeton D. J., Nature, 372:798-781, 1994). Both MEKK1 and MEKK2 are widely expressed in mammalian tissues.
Activation of the JNK pathway has been documented in a number of disease settings, providing the rationale for targeting this pathway for drug discovery. In addition, molecular genetic approaches have validated the pathogenic role of this pathway in several diseases. For example, autoimmune and inflammatory diseases arise from the over-activation of the immune system. Activated immune cells express many genes encoding inflammatory molecules, including cytokines, growth factors, cell surface receptors, cell adhesion molecules, and degradative enzymes. Many of these genes are regulated by the JNK pathway, through activation of the transcription factors AP-1 and ATF-2, including TNFa, IL-2, E-selectin, and matrix metalloproteinases such as collagenase-1 (Manning A. M. and Mercurio F., Exp Opin Invest Drugs, 6: 555-567, 1997). Monocytes, tissue macrophages, and tissue mast cells are key sources of TNFa production. The JNK pathway regulates TNFa production in bacterial lipopolysaccharide-stimulated macrophages, and in mast cells stimulated through the FceRII receptor (Swantek J. L., Cobb M. H., Geppert T. D., Mol. Cell. Biol., 17:6274-6282, 1997; Ishizuka, T., Tereda N., Gerwins, P., Hamelmann E., Oshiba A., Fanger G. R., Johnson G. L., and Gelfiand E. W., Proc. Nat. Acad. Sci. USA, 94:6358-6363, 1997). Inhibition of JNK activation effectively modulates TNFa secretion from these cells. The JNK pathway therefore regulates production of this key pro-inflammatory cytokine. Matrix metalloproteinases (MMPs) promote cartilage and bone erosion in rheumatoid arthritis, and generalized tissue destruction in other autoimmune diseases. Inducible expression of MMPs, including MMP-3 and MMP-9, type II and IV collagenases, are regulated via activation of the JNK pathway and AP-1 (Gum, R., Wang, H., Lengyel, E., Juarez, J., and Boyd, D., Oncogene, 14:1481-1493, 1997). In human rheumatoid synoviocytes activated with TNFa, IL-1, or Fas ligand the JNK pathway is activated (Han Z., Boyle D. L., Aupperle K. R., Bennett B., Manning A. M., Firestein G. S., J. Pharm. Exp. Therap., 291:1-7, 1999; Okamoto K., Fujisawa K., Hasunuma T., Kobata T., Sumida T., and Nishioka K., Arth & Rheum, 40: 919, 1997). Inhibition of JNK activation results in decreased AP-1 activation and collagenase-1 expression (Han et al., supra). The JNK pathway therefore regulates MMP expression in cells involved in rheumatoid arthritis.
Inappropriate activation of T lymphocytes initiates and perpetuates many autoimmune diseases, including asthma, inflammatory bowel disease, and multiple sclerosis. The JNK pathway is activated in T cells by antigen stimulation and CD28 receptor co-stimulation and regulates production of the growth factor IL-2 and cellular proliferation (Su B., Jacinto E., Hibi M., Kallunki T., Karin M., Ben-Neriah Y,. Cell, 77:727-736, 1994; Faris M., Kokot N., Lee L., and Nel A. E., J. Biol. Chem., 271:27366-27373, 1996). Peripheral T cells from mice genetically deficient in JNKK1 show decreased proliferation and IL-2 production after CD28 co-stimulation and PMA/Ca2+ ionophore activation, providing important validation for the role of the JNK pathway in these cells (Nishina H., Bachmann M., Oliveria-dos-Santos A. J., et al., J. Exp. Med., 186: 941-953, 1997). It is known that T cells activated by antigen receptor stimulation in the absence of accessory cell-derived co-stimulatory signals lose the capacity to synthesize IL-2, a state called clonal anergy. This is an important process by which auto-reactive T cell populations are eliminated from the peripheral circulation. Of note, anergic T cells fail to activate the JNK pathway in response to CD3- and CD28-receptor co-stimulation, even though expression of the JNK enzymes is unchanged (Li W., Whaley C. D., Mondino A., and Mueller D. L., Science 271:1272-1276, 1996). Recently, the examination of JNK-deficient mice revealed that the JNK pathway plays a key role in T cell activation and differentiation to T helper 1 and 2 cell types. JNK 1 or JNK2 knockout mice develop normally and are phenotypically unremarkable. Activated naive CD4+T cells from these mice fail to produce IL-2 and do not proliferate well (Sabapathy, K, Hu, Y, K Kallunki, T, Schreiber, M, David, J-P, Jochum, W, Wagner, E, Karin, M,. Curr Biol 9:116-125, 1999). It is possible to induce T cell differentiation in T cells from these mice, generating Th1 cells (producers of IFN-g and TNFβ) and Th2 effector cells (producers of IL-4, IL-5, IL-6, IL-10, and IL-13). Deletion of either JNK1 or JNK2 in mice resulted in a selective defect in the ability of Th1 effector cells to express IFNg. This suggests that JNK1 and JNK2 do not have redundant functions in T cells and that they play different roles in the control of cell growth, differentiation, and death. The JNK pathway therefore, is an important point for regulation of T cell responses to antigen.
Cardiovascular disease (CVD) accounts for nearly one quarter of total annual deaths worldwide. Vascular disorders such as atherosclerosis and restenosis result from dysregulated growth of the vessel wall, restricting blood flow to vital organs. The JNK pathway is activated by atherogenic stimuli and regulates local cytokine and growth factor production in vascular cells (Yang, D D, Conze, D, Whitmarsh, A J, et al., Immunity, 9:575, 1998). In addition, alterations in blood flow, hemodynamic forces, and blood volume lead to JNK activation in vascular endothelium, leading to AP-1 activation and pro-atherosclerotic gene expression (Aspenstrom P., Lindberg U., and Hall A., Curr. Biol. 6:70-77, 1996). Ischemia and ischemia coupled with reperfusion in the heart, kidney, or brain results in cell death and scar formation, which can ultimately lead to congestive heart failure, renal failure, or cerebral dysfunction. In organ transplantation, reperfusion of previously ischemic donor organs results in acute leukocyte-mediated tissue injury and delay of graft function. The JNK pathway is activated by ischemia and reperfusion (Li Y., Shyy J., Li S., Lee J., Su B., Karin M., Chien S., Mol. Cell. Biol., 16:5947-5954, 1996), leading to the activation of JNK-responsive genes and leukocyte-mediated tissue damage. In a number of different settings JNK activation can be either pro- or anti-apoptotic. JNK activation is correlated with enhanced apoptosis in cardiac tissues following ischemia and reperfusion (Pombo C M, Bonventre J V, Avruch J, Woodgett J R, Kyriakis J. M, Force T., J. Biol. Chem. 269:26546-26551, 1994).
Cancer is characterized by uncontrolled growth, proliferation and migration of cells. Cancer is the second leading cause of death with 500,000 deaths and an estimated 1.3 million new cases in the United States in 1996. The role of signal transduction pathways contributing to cell transformation and cancer is a generally accepted concept. The JNK pathway leading to AP-1 appears to play a critical role in cancer. Expression of c-jun is altered in early lung cancer and may mediate growth factor signaling in non-small cell lung cancer (Yin T., Sandhu G., Wolfgang C. D., Burrier A., Webb R. L., Rigel D. F. Hai T., and Whelan J., J. Biol. Chem. 272:19943-19950, 1997). Indeed, over-expression of c-jun in cells results in transformation, and blocking c-jun activity inhibits MCF-7 colony formation (Szabo E., Riffe M., Steinberg S. M., Birrer M. J., Linnnoila R. I., Cancer Res. 56:305-315, 1996). DNA-damaging agents, ionizing radiation, and tumor necrosis factor activate the JNK pathway. In addition to regulating c-jun production and activity, JNK activation can regulate phosphorylation of p53 and, thus, can modulate cell cycle progression (Chen T. K., Smith L. M., Gebhardt D. K., Birrer M. J., Brown P. H,. Mol. Carcinogenesis, 15:215-226, 1996). The oncogene BCR-Ab1, associated with t(9, 22) Philadelphia chromosome translocation of chronic myelogenous leukemia, activates JNK and leads to transformation of hematopoietic cells (Milne D. M., Campbell L. E., Campbell D. G., Meek D. W., J. Biol. Chem. 270:5511-5518, 1995). Selective inhibition of JNK activation by a naturally occurring JNK inhibitory protein, called JIP-1, blocks cellular transformation caused by BCR-Ab1 expression (Raitano A. B., Halpern J. R., Hambuch T. M., Sawyers C. L., Proc. Nat. Acad. Sci USA, 92:11746-11750, 1995). Thus, JNK inhibitors may block transformation and tumor cell growth.
Stroke is the 3rd leading cause of death and a leading cause of disability in the U.S. Stroke, along with neurodegenerative diseases, such as Alzheimer's (AD) and Parkinson's disease (PD) impose a huge burden on the health care industry by impacting the quality of life of those affected. Loss of neuronal cell populations in stroke, AD, or PD underlies the motor and/or cognitive deficiencies in these patient populations. The mechanism by which neurons die in response to insult has not been fully elucidated; however, activation of the JNK pathway has been implicated as a major signaling pathway for neuronal apoptosis. (For review see Mielke K. and Herdegen T. Prog. Neurobiol. 61:45-60, 2000). A variety of insults have been shown to activate the JNK pathway in neurons. For example, activation of JNKs and phosphorylation of c-jun has been shown in brains of rats subjected to axotomy or ischemia with reperfusion, where neuronal cell loss was observed (Herdegen T., Claret F.-X., Kallunki, T., Matin-Villalba A., Winter C., Hunter T. and Karin M. J. Neurosci. 18:5124-5135, 1998). Further, inhibition of the mixed lineage kinase (MLK)-3, an upstream kinase in the JNK pathway, by CEP-1347 prevented motoneuron cell death following growth factor withdrawal in vitro (Maroney A. C., Glicksman M. A., Basma A. N., Walton K. M., Knight Jr. E., Murphy C. A., Bartlett B. A., Finn J. P., Angeles T., Matsuda Y., Neff N. T. and Dionne C. A., J. Neurosci. 18:104-111, 1998), protected cholinergic neurons following excitotoxic injury of the nucleus basalis magnocellularis (Saporito M. S., Brown, E. R., Miller M. S., Murakata C., Neff N. H., Vaught J. L., and Carswell S. Neuroscience 86:461-472, 1998), and blocked the degeneration of midbrain dopamine neurons in mice treated with the neurotoxin, 1-methyl-4-phenyl tetrahydropyridine (Saporito M. S., Brown E. M., Miller M. S. and Carswell S. J. Pharm. Exp. Ther., 1999). While JNK1 and JNK2 enzymes have a widespread tissue distribution, JNK3 is selectively expressed in brain and to a lesser extent in the heart and testis (Dong C., Yang D., Wysk M., Whitmarsh A., Davis R., and Flavell R. Science 270:1-4, 1998). Because of this restricted distribution, JNK3 may be the prevailing kinase mediating neuronal apoptosis. In support of JNK3's involvement in neuronal apoptosis, disruption of the gene encoding JNK3 in mice confers resistance to kainic acid—induced seizures and subsequent hippocampal neuronal cell death (Yang D. D., Kuan C.-Y., Whitmarsh A. J., Rincon M., Zheng T. S., Davis R. J., Rakic P. and Flavell R. A. Nature 389:865-870, 1997). Mounting evidence points to a role for the JNK pathway in neuronal apoptosis. Therefore, selective JNK inhibitors should prevent neuronal cell death observed in disorders and diseases of the CNS.
Accordingly, there is a need in the art for treating or preventing a disease associated with modulation of JNK, compositions comprising modulators of JNK, and methods of modulating JNK and treating or preventing a disorder that is alleviated by modulation of JNK. The present invention fulfills these needs, and provides further related advantages.
Citations or identification of any reference in Section 2 of this application is not to be construed that such reference is prior art to the present application.